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I' , - c! annabinoid Chromatography 101 Chromatography is often called separation science since it ' is a technique for separating compounds in a matrix, In the study presented here we are separating cannabinoids from a matrix of plant material, including chlorophyll, fats, protein and various other related compounds including the essential oils, Cannabinoid chromatography focuses on five compounds: Delta 8 and Delta 9 Tetrahydrocannabinol (/\ 8, 9-THC), Cannibinol (CBN), Cannabidiol (CBD) and Canabinolic Acid (THC-A). The most important being the THC-A in Liquid Phase Chromatography as is the practice here, as opposed to gas chromatography, which is practiced in 99% of other laboratories. The vital difference between gas and liquid phase chromatography is that in gas chromatography the sample is heated to greater than 200 degrees centigrade to perform the analysis in the gas phase, In heating the sample the THC-A is converted 1:1 to /\9- THe. Understanding this principle is key to making oral cannabis medicines. Each of the five cannabinoids has a distinct chemical structure, that is, the carbon, hydrogen and oxygen atoms that make up the different cannabinoids differ in number and spatial arrangement giving them distinct chemical properties, Two of these chemical properties are exploited by LC Chromatography, the first being their retention properties on a silica based, hydrocarbon coated column on which they are separated, Because of the slightly differing chemistries of the individual cannabinoids they will travel along this column at different rates carried by an organic solvent in which they are dissolved, They therefore enter the detector at different times from the beginning of the run, Subsequently, they enter a beam ofUV light, which they absorb, again differently, depending on their chemical structures, This UV absorption is also characteristic of the particular compound and can be used for identification purposes, In order to confirm which cannabinoid is which and how much of each one there is in our test solution we must run pure standards on the Le. These are the individual cannabinoids in pure form made up to a specific known concentration, These standard runs are used to calibrate the Le. That is, we can use these standards to tell the computer the concentration of each peak made by each compound, The peaks are made by the absorption ofUV light by the compound and the area under the peak is directly proportional to the concentration of the test solution, Therefore, knowing the peak area of the standard allows us to calculate the concentration of a sample from its peak area, In summary, by using pure standards ofthe individual cannabinoids we can quantifY cannabinoids in samples and therefore determine their concentration, Furthermore, the pure standards allow us to identifY the individual cannabinoids based on their retention times (time from injection to entering the detector) and their UV spectra.

Cannabinoid Chromatography 101

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Page 1: Cannabinoid Chromatography 101

I' ,

-c!annabinoid Chromatography 101

Chromatography is often called separation science since it 'is a technique for separating compounds in a matrix, In the study presented here we are separating cannabinoids from a matrix of plant material, including chlorophyll, fats, protein and various other related compounds including the essential oils,

Cannabinoid chromatography focuses on five compounds: Delta 8 and Delta 9 Tetrahydrocannabinol (/\8, 9-THC), Cannibinol (CBN), Cannabidiol (CBD) and Canabinolic Acid (THC-A). The most important being the THC-A in Liquid Phase Chromatography as is the practice here, as opposed to gas chromatography, which is practiced in 99% of other laboratories. The vital difference between gas and liquid phase chromatography is that in gas chromatography the sample is heated to greater than 200 degrees centigrade to perform the analysis in the gas phase, In heating the sample the THC-A is converted 1: 1 to /\9-THe. Understanding this principle is key to making oral cannabis medicines.

Each of the five cannabinoids has a distinct chemical structure, that is, the carbon, hydrogen and oxygen atoms that make up the different cannabinoids differ in number and spatial arrangement giving them distinct chemical properties,

Two of these chemical properties are exploited by LC Chromatography, the first being their retention properties on a silica based, hydrocarbon coated column on which they are separated, Because of the slightly differing chemistries of the individual cannabinoids they will travel along this column at different rates carried by an organic solvent in which they are dissolved, They therefore enter the detector at different times from the beginning of the run, Subsequently, they enter a beam ofUV light, which they absorb, again differently, depending on their chemical structures, This UV absorption is also characteristic of the particular compound and can be used for identification purposes,

In order to confirm which cannabinoid is which and how much of each one there is in our test solution we must run pure standards on the Le. These are the individual cannabinoids in pure form made up to a specific known concentration, These standard runs are used to calibrate the Le. That is, we can use these standards to tell the computer the concentration of each peak made by each compound, The peaks are made by the absorption ofUV light by the compound and the area under the peak is directly proportional to the concentration of the test solution, Therefore, knowing the peak area of the standard allows us to calculate the concentration of a sample from its peak area,

In summary, by using pure standards ofthe individual cannabinoids we can quantifY cannabinoids in samples and therefore determine their concentration, Furthermore, the pure standards allow us to identifY the individual cannabinoids based on their retention times (time from injection to entering the detector) and their UV spectra.

Page 2: Cannabinoid Chromatography 101

In the chromatograms on the following pages a few points are in order: The total THC potential refers to the percentage of THC which will become available on heating, for example smoking or baking into brownies. To calc~late THC potential we have to add the % Amount values next to the names /\9-THC and THC-A The total THC potential is then realized. Note that the /\9-THC in unburned samples is often less than 1% and that the THC-A can be as high as 25%* CBN and CBD remain constant on heating. /\8-THC is often present in small amounts and also being psychoactive is included with the /\9-THC, both chromatographically and hence in calculations.

CBD//\9 ratios are provided on a number of chromatograms, this value is given for the 'ground state' cannabis, before it is heated. Obviously, this value will change dramatically after heating the sample, approaching infinity ©. The ground state value is used to provide an indication of the CBD content, since this compound is an important modulator of the /\9-TH C effect.

* We had one sample in our lab that measured out at 25% THC-A and 1 % /\9-THC giving it a total. THC potential of26%. Samples with this level ofTHC potential are rare.

"This will be deducted from your share in paradise". Dr. J. J. Moreau

A Paul Hornby, Ph.D.

June 29, 2003

Cover: 3D Plot of Steve's Budder sample demonstrating different cannabinoids and their chromatographic and UV properties.

Page 3: Cannabinoid Chromatography 101

· Data File C:\HPCHEM\1\DATA\MJ-0408\BD-4 . D Cannabinoids. 100mg/10ml . 2.5 ul.

Sample Name: Budder Green

Injection Date Sample Name Acq. Operator

4 /8/04 1:31:12 PM Budder Green Felipe

Acq. Method C:\HPCHEM\1\METHODS \ MJ-1A.M Last changed 4 / 8/0 4 2:41:05 PM by Felipe

(modified after loading ) Analysis Method C: \ HPCHEM\ 1 \ METHODS \MJ-1A.M Last changed 4/21/04 8:58:23 AM by Felipe

(modified after loading)

Vial o

Inj Volume 2.5 ILl

Method for Cannabinoid Analysis: New Zorbax SBC18 Donated by Tom T. Isocratic. Detection by Diode Array at 219 nm. =========== = =========================================================

DAD1 A, 8ig=219,4 Ref-450,BO (MJ-0408I8D-4.D)

Norm. « 0 0 I

~ Z d; '" 1000 ,0 0

<

,"", ,

~~ ,

<D

500 . N <D ",,"" '" ~N ~~ '" = . '" o~ '"" ~~

N

'JfA~ N <0'" ,J\ :: '" .¢u:I

"":00 ~ ~ NN 0

10 2D 30 40

External Standard Report

g \

50 60

=====================================================================

Sorted By Calib. Data Modified Multiplier Dilution

Retention Time 4 / 21 / 04 8:58 : 19 AM 0.2000

200.0000

Signal 1: DADI A, Sig=219,4 Ref=450,80

RetTime Sig Type Area Amt / Area Amount Grp Name [min] [mAU*s] [%]

-------1--1------1------ ---- 1------- --- 1---------- 1--1---- --- ---- ----15.717 1 VB 4403.82715 2.56423e-5 4 . 51698 CBDA 16.541 1 VB 1126.55017 2.61517e-5 1 . 17845 CBD 26.317 1 BBA 713.08832 2.56423e-5 7.31410e - 1 CBN 36.736 1 BBA 1.12777e4 2.56423e-5 11.56747 -9-THC 50.889 1 BBA 8.7864ge4 2.56423e-5 90.12243 THC-A

Totals : 108.11673

Results obtained with enhanced integratorl 1 Warnings or Errors :

Warning : Calibration warnings (see calibration table listing)

*** End o f Report ***

Instrument 1 4/21/04 8:59:23 AM Felipe Page 1 of 1

mi

Page 4: Cannabinoid Chromatography 101

Data File C:\HPCHEM\1 \ DATA\ MJ-0408 \ BD-4 . D Sample Name: Budder Green

Purity results peak 15 at 36 .736 min.

1 AD A S · 2 9 f o 80 (MJ 0 08\ 19na D 1 , 19= 1 ,4 Re =45 , - 4 BD-4.D mAU

1-·

1400 I I I I

I 1200 c I I·

1000 c I I I I

800 I I I I

600 I I I I

400 I I

.t iAi 200 I I

0 I I

10 20 30 40 50 60 mi

I I

250 300 :J5o 400 450 500 550 nrr 36 37 38 mi

-> The purity factor is within the threshold limit. <-

Purity factor

Threshold Reference

Spectra

999 .362 (3 of 3 spectra are within the threshold limit. ) 970.000 (Set by user ) Peak start and end spectra (integrated) (35.782 / 37.781) 3 (Selection automatic, 3)

*** End of Report ***

Instrument 1 4/21/04 9:00 : 35 AM Felipe Page 1 of 1

Page 5: Cannabinoid Chromatography 101

Data File C:\HPCHEM\ 1\DATA\MJ-0413\BD-1.D Cannabinoids.

Sample Name: BD 1k ppm

Injection Date Sample Name Acq. Operator

4/13/04 6:19:03 PM BD 1k ppm Felipe

Acq. Method C:\HPCHEM\l\METHODS\MJ-IA.M Last changed 4 / 13 /0 4 7 : 24:03 PM by Felipe

(modified after loading) Analysis Method C: \ HPCHEM\ 1\METHODS \ MJ-1A.M Last changed 4 / 21 / 04 8:54:37 AM by Felipe

(modified after loading)

Vial o

Inj Volume 5 Jil

Method for Cannabinoid Analysis: New Zorbax SBC18 Donated by Tom T. Isocratic. Detection by Diode Array at 219 nm.

DADl A, 5ig=219,4 Ref=450,5O (MJ-0413IBD·1 .D)

Norm. c « 0

~ 0 I

400 alO Z I-Oal '" 2>

300 c ,0 0

ZOO c !If -'" N -A ~ a ...,'" '" , ~ 100 ~

..,~ ~ 0=; :A:! 0

N"/FI <Xl - N

10 20 30 40 50 60

Area Percent Report =====================================================================

Sorted By Retention Time Calib. Data Modified Multiplier

Wednesday, April 21, 2004 8:53:44 AM 1.0000

Dilution 1. 0000

Signal 1: DAD1 A, Sig=219,4 Ref=450,80

Peak RetTime Sig Type Area Area Name # [min] [mAU*s] .. o

----1-------1---1------1-- --- -----1--------1------------------ -- --- --1 13 . 711 1 PBA 1041 . 45386 3.9194 CBDA 2 14 .679 1 PBA 266.85358 1.0043 CBD 3 21.572 1 PBA 135.87231 0.5113 CBN 4 29.611 1 BBA 2865.33057 10.7835 A9-THC 5 39.338 1 BBA 2 . 17004e4 81.6683 THC-A

Totals : 2.60100e4

Results obtained with enhanced integrator!

*** End of Report ***

, Instrument 1 4 /2 1/04 8:54:37 AM Felipe Page 1 of 1

mi

Page 6: Cannabinoid Chromatography 101

Data File C:\HPCHEM\1\DATA\ MJ-0408\BD-4.D Sample Name: Budder Green

purity results peak 16 at 50.889 min.

1 S · 2 9 f o 80 (MJ 0408 \ BD D) S~gna DADl A, ~q= 1 ,4 Re =45 , - -4. mAU _

~-,

1400 -I I I I

1200 I I I I

1000 I I I I

800 I I I

600 I I

400 I

,l, 1\ I

200 1 0 \i

10 20 30 40 50 60 mi

250 300 350 400 450 500 550 n 50 52 mi

-> The purity factor is within the threshold limit. <-

purity factor

Thresho ld Reference

Spectra Warning

999.920 (3 of 3 spectra are within the threshold limit.) 970.000 (Set by user ) Peak start and end spectra (integrated) (49.714 / 52.718) 3 (Selection automatic, 3) Spectral absorbances > 1000 rnAU (see help for more information)

*** End of Report ***

Instrument 1 4/21/04 8:58:38 AM Felipe Page 1 of 1

Page 7: Cannabinoid Chromatography 101

Mariceuticals Natural Medications Attention: Steve

April 29, 2004

Budder

Certificate of Analysis

Parameter Tested PesticidelHerbicide Screen

nd = not detected (detection llffilt I ppm).

Methodology: HPLCIDAD

Thank you for using AA

Dr. 0

Value (ppm) nd <1.0

Page 8: Cannabinoid Chromatography 101

Certificate of Analysis

Number: 03-S-44

To: Steve

Sample: "Budder"

Results of analysis:

(I) Plate Count (cfu/g) (2) Total colifonns (MPN/g) (3) Fecal colifonns (MPN/g) (2) E. coli !lOg (3) Staphylococcus aureus (cfu/g) (4) Salmonella/25g (5) Yeast (cfu/g) (5) Mold (cfu/g)

cfu - colony forming unit MPN - Most Probable Number ND - not detected

Methodology:

Date of Report: May 3, 2004

Submitted: April 27, 2003

1000 <3 (ND) <3 (ND) <3 (ND) < 10 Negative <4 <4

Analyses were conducted on a day of arrival according to: Compendium of Analytical Methods. HPB Methods of Microbiological Analysis of Food, (I) - MFHPB-18, (2) ­MFHPB-19, (3) - MFHPB-21, (4) -MFHPB-20, (5) -MFHPB-22,

Thank you for using AA

Dr. 0

Page 9: Cannabinoid Chromatography 101

' Data File C:\HPCHEM\1\DATA\PES-0428\BD-2.D Sample Name: Budder

Pesticide/Herbicide Screen.

Injection Date Sample Name Acq. Operator

4 / 28 / 04 1:2 8 :57 PM Budder Felipe

Vial o

Method C:\HPCHEM\ 1 \ METHODS \ PEST- 1B.M Inj Volume 10 pI

Last changed 5/ 2/ 04 11:12:00 AM by Felipe Method for detection o f Pesitcides / Herbicides uses C18 column and P04 aqueous an d '1 h acetonltrl e organlc PJ ases. Detectlon at 245 nm .

DADl A, Sig-245,4 Ref- 450,80 (PES-0428\8D-2.D)

mAU ~" 1~

10

5

0 _l

-5

-10

10 20 30 40

===================================================================== External Standard Report

================================== === = ====== = == = =====================

Sorted By Calib. Data Modified Multiplier Dilution

Signal 1: Signal 2: Signal 3:

DAD 1 NEW, NEW,

A, Sig=245,4 not found not found

Retention Time 5/2/04 11:11:48 AM 1.0000 1. 0000

Ref=450,80

Ret Time Sig Type Area Amt / Area Amount Grp Name [min] [ppm]

--- - ---/--/------/----------/----------/----------/--/---------------7.000 1 Dirun

50

7.200 1 Desethylatrazine 8 . 100 1 Benzthiazuron B.400 1 Hexazinon 9.200 1 Metoxuron 9 _ 500 1 Simazine 9 . 600 1 Methabenzthiazuron 9.700 1 Nonlinuron 9.730 1 Atrazine 9. BOO 1 Isoproturon

11.600 1 Metobromuron 12 . 000 1 Metzachlor 12 . 600 1 Propazine 14 . 600 1 Setbutylazine 15.600 1 Tertbutylazine 17 . 900 1 Linuron 19 . 400 1 Metalochlor

Instrument 1 4/ 28 / 04 2:44:32 PM Felipe Page 1 of 4

mi

Page 10: Cannabinoid Chromatography 101

Data File C:\HPCHEM\1\DATA\PES-0428\BD-2.D Sample Name: Budder

RetTime Sig Type Area Amt/Area Amount Grp Name [min) [ppm)

-------1-- 1------1----------1----------1----------1--1---------------20.100 1 Avid

Totals : 0.00000

Signal 1: DAD1 A, Sig=245,4 Ref=450,80 Unca1ibrated Peaks compound name not specified Signal 2: NEW, not found Uncalibrated Peaks compound name not specified Signal 3: NEW, not found Uncalibrated Peaks compound name not .specified

. RetTime Sig Type Area Amt/Area Amount Grp Name [min) [ppm)

-- ---- -1--1------1--- ----- --1----------1-- ------- -1--1- --------------4.934 1 PBA 68.61839 0.00000 0.00000 ? 5.767 1 PBA 233.40512 0 . 00000 0.00000 ?

Uncalib. totals

Results obtained with enhanced integrator! 2 warnings or Errors :

0.00000

Warning Calibration warnings (see calibration table listing) Warning : Calibrated compound(s) not found

===================================================================== =====================================================================

Area Percent Report =====================================================================

Sorted By Calib . Data Modified Multiplier Dilution

Retention Time 5/2/04 11:11:48 AM 1.0000 1.0000

Signal 1: DAD1 A, Sig=245,4 Ref=450,80

Peak Ret Time Sig Type Area Area Name # [min] %

----1------ -1--- 1------ 1----------1--------1- ------------------------1 0.000 2 0.00000 0.0000 Avid 2 0 . 000 3 0.00000 0.0000 Avid 3 0.000 2 0.00000 0.0000 Metalochlor 4 0.000 3 0.00000 0.0000 Metalochlor 5 0.000 2 0.00000 0.0000 Linuron 6 0.000 3 0.00000 0.0000 Linuron 7 0.000 2 0.00000 0.0000 Tertbutylazine 8 0.000 3 0.00000 0 . 0000 Tertbutylazine 9 0.000 2 0 . 00000 0 . 0000 Setbutylazine

10 0.000 3 0.00000 0.0000 Setbuty lazine 11 0 . 000 2 0.00000 0.0000 Propazine 12 0.000 3 0.00000 0.0000 Propazine 13 0.000 2 0.00000 0.0000 Metzachlor 14 0.000 3 0.00000 0.0000 Metzachlor 15 0.000 2 0.00000 0.0000 Metobromuron

Instrument 1 4/28/04 2:44:32 PM Felipe Page 2 of 4

Page 11: Cannabinoid Chromatography 101

Data File C:\HPCHEM\1\DATA\PES-0428\BD-2 . D Sample Name: Budder

Peak RetTime Sig Type Area Area Name # [min] %

----1-------1---1------1----7-----1--------1-------------------------16 0.000 3 0.00000 0.0000 Metobromuron 17 0.000 2 0.00000 0.0000 Isoproturon 18 0 . 000 3 0.00000 0.0000 Isoproturon 19 0.000 2 0.00000 0.0000 Atrazine 20 0 . 000 3 0.00000 0.0000 Atrazine 21 0 . 000 2 0 . 00000 0 . 0000 Nonlinuron 22 0 . 000 3 0.00000 0.0000 Nonlinuron 23 0 . 000 2 0 . 00000 0 . 0000 Methabenzthiazuron 24 0.000 3 0.00000 0.0000 Methabenzthiazuron 25 0.000 2 0.00000 0.0000 Simazine 26 0 . 000 3 0.00000 0 . 0000 Simazine 27 0.000 2 0.00000 0.0000 Metoxuron 28 0 . 000 3 0.00000 0.0000 Metoxuron 29 0 . 000 2 0.00000 0.0000 Hexazinon 30 0.000 3 0.00000 0 . 0000 Hexazinon 31 0.000 2 0.00000 0 . 0000 Benzthiazuron 32 0.000 3 0.00000 0.0000 Benzthiazuron 33 0.000 2 0.00000 0.0000 Desethylatrazine 34 0.000 3 0.00000 0.0000 Desethylatrazine 35 0.000 2 0 . 00000 0.0000 Dirun 36 0 . 000 3 0 . 00000 0 . 0000 Dirun 37 7.000 1 0.00000 0.0000 Dirun 38 7 . 200 1 0.00000 0.0000 Desethy1atrazine 39 8.100 1 0.00000 0.0000 Benzthiazuron 40 8.400 1 0.00000 0 . 0000 Hexazinon 41 9.200 1 0.00000 0.0000 Metoxuron 42 9.500 1 0 . 00000 0.0000 Simazine 43 9.600 1 0.00000 0.0000 Methabenzthiazuron 44 9 . 700 1 0 '. 00000 0.0000 Nonlinuron 45 9.730 1 0 . 00000 0.0000 Atrazine 46 9.800 1 0.00000 0 . 0000 Isoproturon 47 11.600 1 0.00000 0 . 0000 Metobromuron 48 12 . 000 1 0.00000 0.0000 Metzachlor 49 12.600 1 0.00000 0 . 0000 Propazine 50 14 . 600 1 0 . 00000 0.0000 Setbutylazine 51 15.600 1 0 . 00000 0.0000 Tertbutylazine 52 17.900 1 0 . 00000 0.0000 Linuron 53 19.400 1 0 . 00000 0 . 0000 Metalochlor 54 20.100 1 0 . 00000 0.0000 Avid

Totals : 0.00000

Uncalibrated Peaks: Peak RetTime Sig Type Area Area Name

# [min] % ----1-------1---1------1----------1--------1-------------------------

1 4.934 1 PBA 68.61839 22.7196? 2 5 . 767 1 PBA 233.40512 77.2804?

Uncalib . totals 302.02351

Results obtained with enhanced integrator! 2 Warnings or Errors :

Instrument 1 4/28/04 2:44:32 PM Felipe Page 3 of 4

Page 12: Cannabinoid Chromatography 101

" Data File C:\HPCHEM\1\DATA\PES-0428\BD-2.D Sample Name: Budder

, Warning Warning

Calibration warnings (see calibration table listing) Calibrated compound{s) not found

===================================================================== *** End of Report ***

Instrumeilt 1 4/28/04 2:44:32 PM Felipe Page 4 of 4